Role of subsite +1 residues in pH dependence and catalytic activity of the glycoside hydrolase family 1 beta-glucosidase BGL1A from the basidiomycete Phanerochaete chrysosporium

The basidiomycete Phanerochaete chrysosporium produces two glycoside hydrolase family 1 intracellular beta-glucosidases, BGL1A and BGL1B, during the course of cellulose degradation. In order to clarify the catalytic difference between two enzymes, in spite of their high similarity in amino acid sequences (65%), five amino acids around the catalytic site of BGL1A were individually mutated to those of BGL1B (V173C, M177L, D229N, H231D, and K253A), and the effects of the mutations on cellobiose hydrolysis were evaluated. When the kinetic parameters (K(m) and k(cat)) were compared at the optimum pH for the wild-type enzyme, the kinetic efficiency was decreased in the cases of D229N, H231D, and K253A, but not V173C or M177L. The pH dependence of cellobiose hydrolysis showed a significantly more acidic pH profile for the D229N mutant, compared with the wild-type enzyme. Since D229 is located between K253 and the putative acid/base catalyst E170, we prepared the double mutant D229N/K253A, and found that its hydrolytic activity at neutral pH was restored to that of the wild-type enzyme. Our results indicate that the interaction between D229 and K253 is critical for the pH dependence and catalytic activity of BGL1A. Biotechnol. Bioeng.     

Enhancement of the enzymatic activity of ribonuclease HI from Thermus thermophilus HB8 with a suppressor mutation method

A genetic method for isolating a mutant enzyme of ribonuclease HI (RNase HI) from Thermus thermophilus HB8 with enhanced activity at moderate temperatures was developed. T. thermophilus RNase HI has an ability to complement the RNase H-dependent temperature-sensitive (ts) growth phenotype of Escherichia coli MIC3001. However, this complementation ability was greatly reduced by replacing Asp(134), which is one of the active site residues, with His, probably due to a reduction in the catalytic activity. Random mutagenesis of the gene encoding the resultant D134H enzyme, followed by screening for second-site revertants, allowed us to isolate three single mutations (Ala(12) --> Ser, Lys(75) --> Met, and Ala(77) --> Pro) that restore the normal complementation ability to the D134H enzyme. These mutations were individually or simultaneously introduced into the wild-type enzyme, and the kinetic parameters of the resultant mutant enzymes for the hydrolysis of a DNA-RNA-DNA/DNA substrate were determined at 30 degrees C. Each mutation increased the k(cat)/K(m) value of the wild-type enzyme by 2.1-4.8-fold. The effects of the mutations on the enzymatic activity were roughly cumulative, and the combination of these three mutations increased the k(cat)/K(m) value of the wild-type enzyme by 40-fold (5.5-fold in k(cat)). Measurement of thermal stability of the mutant enzymes with circular dichroism spectroscopy in the presence of 1 M guanidine hydrochloride and 1 mM dithiothreitol showed that the T(m) value of the triple mutant enzyme, in which all three mutations were combined, was comparable to that of the wild-type enzyme (75.0 vs 77.4 degrees C). These results demonstrate that the activity of a thermophilic enzyme can be improved without a cost of protein stability.     

Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant

Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively. The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity

The catalytic reaction mechanism and binding of substrates was investigated for the multisubstrate Drosophila melanogaster deoxyribonucleoside kinase. Mutation of E52 to D, Q and H plus mutations of R105 to K and H were performed to investigate the proposed catalytic reaction mechanism, in which E52 acts as an initiating base and R105 is thought to stabilize the transition state of the reaction. Mutant enzymes (E52D, E52H and R105H) showed a markedly decreased k(cat), while the catalytic activity of E52Q and R105K was abolished. The E52D mutant was crystallized with its feedback inhibitor dTTP. The backbone conformation remained unchanged, and coordination between D52 and the dTTP-Mg complex was observed. The observed decrease in k(cat) for E52D was most likely due to an increased distance between the catalytic carboxyl group and 5'-OH of deoxythymidine (dThd) or deoxycytidine (dCyd). Mutation of Q81 to N and Y70 to W was carried out to investigate substrate binding. The mutations primarily affected the K(m) values, whereas the k(cat) values were of the same magnitude as for the wild-type. The Y70W mutation made the enzyme lose activity towards purines and negative cooperativity towards dThd and dCyd was observed. The Q81N mutation showed a 200- and 100-fold increase in K(m), whereas k(cat) was decreased five- and twofold for dThd and dCyd, respectively, supporting a role in substrate binding. These observations give insight into the mechanisms of substrate binding and catalysis, which is important for developing novel suicide genes and drugs for use in gene therapy.     

The role of Glu(192) in the allosteric control of the S(2)' and S(3)' subsites of thrombin

Thrombin is an allosteric protease controlled through exosites flanking the catalytic groove. Binding of a peptide derived from hirudin (Hir(52-65)) and/or of heparin to these opposing exosites alters catalysis. We have investigated the contribution of subsites S(2)' and S(3)' to this allosteric transition by comparing the hydrolysis of two sets of fluorescence-quenched substrates having all natural amino acids at positions P(2)' and P(3)'. Regardless of the amino acids, Hir(52-65) decreased, and heparin increased the k(cat)/K(m) value of hydrolysis by thrombin. Several lines of evidence have suggested that Glu(192) participates in this modulation. We have examined the role of Glu(192) by comparing the catalytic activity of thrombin and its E192Q mutant. Mutation substantially diminishes the selectivity of thrombin. The substrate with the "best" P(2)' residue was cleaved with a k(cat)/K(m) value only 49 times higher than the one having the "least favorable" P(2)' residue (versus 636-fold with thrombin). Mutant E192Q also lost the strong preference of thrombin for positively charged P(3)' residues and its strong aversion for negatively charged P(3)' residues. Furthermore, both Hir(52-65) and heparin increased the k(cat)/K(m) value of substrate hydrolysis. We conclude that Glu(192) is critical for the P(2)' and P(3)' specificities of thrombin and for the allostery mediated through exosite 1.     

Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site

Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.     

Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of Par-1 on intact platelets

The activation of human platelets by alpha-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ibalpha also has a high affinity binding site for alpha-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIbalpha may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by alpha-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k(cat)/K(m) value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 +/- 0.1 x 10(7) m(-1) sec(-1). The hydrolysis rate of PAR-1 by thrombin was measured under conditions in which thrombin binding to GpIb was inhibited by different strategies, with the following results. 1) Elimination of GpIbalpha on platelet membranes by mocarhagin treatment reduced the k(cat)/K(m) value by about 6-fold. 2) A monoclonal anti-GpIb antibody reduced the apparent k(cat)/K(m) value by about 5-fold. 3) An oligonucleotide DNA aptamer, HD22, which binds to the thrombin heparin-binding site (HBS) and inhibits thrombin interaction with GpIbalpha, reduced the apparent k(cat)/K(m) value by about 5-fold. 4) Displacement of alpha-thrombin from the binding site on GpIb using PPACK-thrombin reduced the apparent k(cat)/K(m) value by about 5-fold, and 5) mutation at the HBS of thrombin (R98A) caused a 5-fold reduction of the apparent k(cat)/K(m) value of PAR-1 hydrolysis. Altogether these results show that thrombin interaction with GpIb enhances the specificity of thrombin cleavage of PAR-1 on intact platelets, suggesting that GpIb may function as a "cofactor" for PAR-1 activation by thrombin.     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

Synthesis of long-chain chitooligosaccharides by a hypertransglycosylating processive endochitinase of Serratia proteamaculans 568

We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40 °C, where it showed a K(m) of 83 mg ml(-1), a k(cat) of 3.9 × 10(2) h(-1), and a k(cat)/K(m) of 4.7 h mg(-1) ml(-1) on colloidal chitin. On chitobiose, the K(m), k(cat), and k(cat)/K(m) were 203 μM, 1.3 × 10(2) h(-1), and 0.62 h(-1) μM(-1), respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.     

Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.     

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.     

A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad

The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.     

Beta-glycosidase of Thermus thermophilus KNOUC202: gene and biochemical properties of the enzyme expressed in Escherichia coli

The beta-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202/beta-gly) was composed of 1296 bp encoding a beta-glycosidase (KNOUC202beta-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. coli JM109(DE3). The enzyme hydrolyzed beta-glycosides whose glycone are galactose, glucose and fucose well, however showed no or very low activity on beta-D-glycosides whose glycone are disaccharides and xylose. kcat of the enzyme for the hydrolysis of p-Nph-beta-D-Glcp was lower than those for p-Nph-beta-D-Galp and ONPG, however K(m) for p-Nph-beta-D-Glcp was highly lower than those for p-Nph-beta-D-Galp and ONPG resulting in the catalytic efficiency(k(cat)/K(m)) for the hydrolysis of p-Nph-beta-D-Glcp much higher than those for p-Nph-beta-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90 degrees C. The enzyme has high thermostability, not losing its activity at 80 degrees C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T(m) of 100.0 +/- 0.031 degrees C in 0.02 M Tris-HCl buffer of pH 8.2. The beta-glycosidase produced a disaccharide composed of galactose as transglycosylation byproduct during hydrolysis of lactose.     

Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.     

Kinetic and spectroscopic characterization of the H178A methionyl aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. The effect of altering H178 on activity is, in general, due to a decrease in k(cat). The k(cat) value for Co(II)-loaded H178A decreased 70-fold toward MGMM and 290-fold toward MP-p-NA compared to the WT enzyme, while k(cat) decreased 50-fold toward MGMM for the Fe(II)-loaded H178A enzyme and 140-fold toward MP-p-NA. The K(m) values for MGMM remained unaffected, while those for MP-p-NA increased approximately 2-fold for Co(II)- and Fe(II)-loaded H178A. The k(cat)/K(m) values for both Co(II)- and Fe(II)-loaded H178A toward both substrates ranged from approximately 50- to 580-fold reduction. The pH dependence of log K(m), log k(cat), and log(k(cat)/K(m)) of both WT and H178A EcMetAP-I were also obtained and are identical, within error, for H178A and WT EcMetAP-I. Therefore, H178A is catalytically important but is not required for catalysis. Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.     

Kinetic and spectroscopic analysis of the catalytic role of H79 in the methionine aminopeptidase from Escherichia coli

To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli ( EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in k cat. Interestingly, the K m value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, k cat/ K m values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded H79A EcMetAP-I decreased by approximately 2.6-fold while k cat decreased by approximately 3.5-fold. The observed K m value for Mn(II)-loaded H79A EcMetAP-I was approximately 1.4-fold larger than that observed for WT EcMetAP-I, resulting in a k cat/ K m value that is lower by approximately 3.4-fold. Metal binding, UV-vis, and EPR data indicate that the active site is unperturbed by mutation of H79, as suggested by X-ray crystallographic data. Kinetic isotope data indicate that H79 does not transfer a proton to the newly forming amine since a single proton is transferred in the transition state for both the WT and H79A EcMetAP-I enzymes. Therefore, H79 functions to position the substrate by hydrogen bonding to either the amine group of the peptide linkage or a backbone carbonyl group. Together, these data provide new insight into the catalytic mechanism of EcMetAP-I.     

Engineering the substrate specificity of Alcaligenes D-aminoacylase useful for the production of D-amino acids by optical resolution

D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of D-amino acid from the racemic mixture of N-acyl-DL-amino acids. However, its substrate specificity is biased toward certain N-acyl-D-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other D-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k(cat)/K(m)) of mutant F191W toward N-acetyl-D-Trp and N-acetyl-D-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k(cat)/K(m)) of mutant L298A toward N-acetyl-D-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of D-Trp which is an important building block of some therapeutic drugs.     

Enhancement, relaxation, and reversal of the stereoselectivity for phosphotriesterase by rational evolution of active site residues

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.     

Cold adaptation of xylose isomerase from Thermus thermophilus through random PCR mutagenesis. Gene cloning and protein characterization.

Random PCR mutagenesis was applied to the Thermus thermophilus xylA gene encoding xylose isomerase. Three cold-adapted mutants were isolated with the following amino-acid substitutions: E372G, V379A (M-1021), E372G, F163L (M-1024) and E372G (M-1026). The wild-type and mutated xylA genes were cloned and expressed in Escherichia coli HB101 using the vector pGEM-T Easy, and their physicochemical and catalytic properties were determined. The optimum pH for xylose isomerization activity for the mutants was approximately 7.0, which is similar to the wild-type enzyme. Compared with the wild-type, the mutants were active over a broader pH range. The mutants exhibited up to nine times higher catalytic rate constants (k(cat)) for d-xylose compared with the wild-type enzyme at 60 degrees C, but they did not show any increase in catalytic efficiency (k(cat)/K(m)). For d-glucose, both the k(cat) and the k(cat)/K(m) values for the mutants were increased compared with the wild-type enzyme. Furthermore, the mutant enzymes exhibited up to 255 times higher inhibition constants (K(i)) for xylitol than the wild-type, indicating that they are less inhibited by xylitol. The thermal stability of the mutated enzymes was poorer than that of the wild-type enzyme. The results are discussed in terms of increased molecular flexibility of the mutant enzymes at low temperatures.